Technical advances in identification, cloning, expression and manipulation of nucleic acid molecules and deciphering of the human genome have greatly accelerated discovery of novel therapeutics based upon deciphering of the human genome. Rapid nucleic acid sequencing techniques can now generate sequence information at unprecedented rates and, coupled with computational analyses, allow the assembly of overlapping sequences into the partial and entire genomes as well as identification of polypeptide-encoding regions. A comparison of a predicted amino acid sequence against a database compilation of known amino acid sequences allows one to determine the extent of homology to previously identified sequences and/or structural landmarks. The cloning and expression of a polypeptide-encoding region of a nucleic acid molecule provides a polypeptide product for structural and functional analyses. The manipulation of nucleic acid molecules and encoded polypeptides to create variant and derivatives thereof may confer advantageous properties on a product for use as a therapeutic.
In spite of significant technical advances in genome research over the past decade, the potential for development of novel therapeutics based on the human genome is still largely unrealized. Many genes encoding potentially beneficial polypeptide therapeutics or those encoding polypeptides, which may act as “targets” for therapeutic molecules, have still not been identified.
Accordingly, it is an object of the invention to identify novel polypeptides, and nucleic acid molecules encoding the same, which have diagnostic or therapeutic benefit.
After years of study in necrosis of tumors, tumor necrosis factors (TNFs) α and β were finally cloned in 1984. The ensuing years witnessed the emergence of a superfamily of TNF cytokines, including fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD40 ligand (CD40L), TNF-related apoptosis-inducing ligand (TRAIL, also designated AGP-1), osteoprotegerin binding protein (OPG-BP or OPG ligand), 4-1BB ligand, LIGHT, APRIL, and TALL-1. Smith et al. (1994), Cell, 76: 959–962; Lacey et al. (1998), Cell, 93: 165–176; Chichepotiche et al. (1997), J. Biol. Chem., 272: 32401–32410; Mauri et al. (1998), Immunity, 8: 21–30; Hahne et al. (1998), J. Exp. Med., 188: 1185–90; Shu et al. (1999), J. Leukocyte Biology, 65: 680–3. This family is unified by its structure, particularly at the C-terminus. In addition, most members known to date are expressed in immune compartments, although some members are also expressed in other tissues or organs, as well. Smith et al. (1994), Cell 76: 959–62. All ligand members, with the exception of LT-α, are type II transmembrane proteins, characterized by a conserved 150 amino acid region within C-terminal extracellular domain. Though restricted to only 20–25% identity, the conserved 150 amino acid domain folds into a characteristic β-pleated sheet sandwich and trimerizes. This conserved region can be proteolyticaly released, thus generating a soluble functional form. Banner et al. (1993), Cell, 73: 431–445.
Many members within this ligand family are expressed in lymphoid enriched tissues and play important roles in the immune system development and modulation. Smith et al. (1994). For example, TNFα is mainly synthesized by macrophages and is an important mediator for inflammatory responses and immune defenses. Tracey & Cerami (1994), Annu. Rev. Med., 45: 491–503. Fas-L, predominantly expressed in activated T cell, modulates TCR-mediated apoptosis of thymocyts. Nagata et al. (1995) Immunology Today, 16:39–43; Castrim et al. (1996), Immunity, 5:617–27. CD40L, also expressed by activated T cells, provides an essential signal for B cell survival, proliferation and immunoglobulin isotype switching. Noelle (1996), Immunity, 4: 415–9.
The cognate receptors for most of the TNF ligand family members have been identified. These receptors share characteristic multiple cysteine-rich repeats within their extracellular domains, and do not possess catalytic motifs within cytoplasmic regions. Smith et al. (1994). Two subgroups of TNFR homologues: Fas, TNFR1, DR3, DR4, DR5, and DR6 contains intracellular death domain which bind TRAD or FADD. This leads to activation of caspase 8 and apoptosis. Locksley et al. (2001) Cell 104: 487–501. However, signaling through death-receptors can also be required for proliferation of hepatocytes and T cells. Strasser et al., (1999) Intl. J. Biochem. Cell Biol. 31: 533–537, Yamada et al. (1997), Proc. Natl. Acad. Of Sci. U.S.A, 94: 1441–6.. The other group including TNFR2, CD40, or CD30 bind TNF-Receptor Associated Factors (TRAFs), molecular adapters that couple these surface receptors to downstream signaling cascades. This leads to activation of JNK and NFKB which can promote cell growth and survival. These proteins therefore play critical roles in morphogenesis, the control of apoptosis, differentiation, or proliferation. TNF/TNFR superfamily proteins are now extensively studied as targets for therapies against many human diseases such as atherosclerosis, allograft rejection, arthritis, and cancer. Locksley et al. (2001), Williams et al. (2000), Ann. Rhem. Dis. 59: i75–80.
In addition to the membrane associated receptor molecules described above, a number the receptors belonging to the TNF-receptor supergene family exist as soluble ligand binding proteins. Many of the soluble forms of the transmembrane receptors were subsequently identified as containing only the extracellular ligand binding domain(s) of the receptors. For example, a soluble form of TNF receptor has been found in urine and serum (see U.S. Pat. No. 5,843,789 and Nophar et al., EMBO J., 9(10):3269–3278, 1990), and have been shown to arise by proteolytic cleavage of cell surface TNF-receptors (Wallach et al., Agents Actions Suppl., 35:51–57, 1991). These soluble forms of receptor molecules have been implicated in the modulation of TNF activity by not only interfering with TNF binding to its receptor, but also by stabilizing the TNF structure and preserving its activity, thus prolonging some of its effects (Aderka et al, Cytokine & Growth Factor Reviews, 7(3):231–240, 1996).
Members of the tumor necrosis factor superfamilies of ligands and cell-surface receptors regulate immune function and most TNF/TNFR superfamily proteins, such as FASL/FAS, CD40L/CD40, TNF/TNFR, or LTβ/LTβR to name a few, are expressed in the immune system, where the coordinate immune cell homeostasis, activation induced cell death, T cells priming, functions and survival of dendritic cells, or the formation of germinal centers and lymphoid organs such as Peyer's patches and lymph nodes. Fu et al. (1999), Ann. Rev. Immunol. 17: 399–433, Grewal et al. (1998), Ann. Rev. Immunol. 166: 111–135. Recently, novel members of this large families have been identified that have critical functions in immunity and couple lymphoid cells with other organ systems such as bone morphogenesis and mammary gland formation in pregnancy.
Because of the crucial role that members of the TNF family of ligands and their receptors (membrane-associated and soluble) play in the immunological system and in a variety of disease processes, a need exists to identify and characterize novel members of these families, for use to improve diagnosis and therapy.